The overall goal of our research is to understand the origin, development and function of embryonic Natural Killer (NK) cells. NK cells are innate immune system lymphocytes and a current target population for cell- based adoptive immunotherapies. However, NK cell therapy has been demonstrated to be effective in only a subset of patients, possibly due in part to the highly heterogeneous nature of NK cell subsets found across donors. In contrast, the generation of NK cells from human pluripotent stem cells (hPSCs) is a particularly exciting alternative, as it would eliminate genetic variability across donor sources, ensuring highly reproducible generation of anti-tumor NK cells. While NK cells in the adult are derived from hematopoietic stem cell (HSC)- derived lymphoid progenitors, the developmental origin of NK cells remains poorly understood. We have demonstrated that HSC-independent hematopoiesis occurs in multiple waves in the early murine embryo, including multipotential ?erythro-myeloid progenitors? (EMP). Similarly, we have developed an in vitro hPSC stage-specific differentiation approach that recapitulates many aspects of embryonic hematopoietic development. Combining these systems, our preliminary parallel studies indicate that HSC-independent, ?extra-embryonic? erythro-myeloid progenitors can generate NK cells, which are morphologically and functionally distinct from ?intra-embryonic? hPSC-derived definitive progenitors, or neonatal donors. The focus of this proposal is to bring together expertise in mouse developmental biology and hPSC directed differentiation, as complementary model systems, to identify the origin(s) of embryonic NK cells, and their translational potential. We hypothesize that these fetal NK cells are a clinically-relevant source of NK cells, with robust antitumor capacity. We will test this hypothesis across three Specific Aims. In Aim 1, we will characterize the ontogenic origins of murine embryonic and hPSC-derived NK cells, establishing their lineage specification from a multipotential myeloid progenitor and the transcriptional regulatory programs. In Aim 2, we will establish the contribution of embryonic NK cells to the adult lymphocyte repertoire, and functionally characterize the adoptive transfer antitumor potential of HSC-independent NK cells. The successful completion of these studies will provide us with a more comprehensive understanding of mammalian hematopoietic development. This is of fundamental importance to our understanding of the development of the innate immune system and the in vitro generation of NK cells for a wide array of regenerative medicine applications, including anti-cancer therapy.